Ribbon synapses transmit sensory information in the visual, auditory, and vestibular systems and are essential for the function of these fundamental senses. These specialized synapses derive their name from a complex organelle, the synaptic ribbon, which is embedded in the cytomatrix at the presynaptic active zone of sensory neurons. When ribbons are disrupted, vision and hearing are strongly impaired, because ribbons are required for faithful transmission of sensory signals in the retina and inner ear. To carry out thi task, ribbons must support both fast, transient and slower, sustained release of neurotransmitter, which they accomplish by enhancing the size and accessibility of the readily releasable pool of synaptic vesicles. Ribbons tether large numbers of synaptic vesicles to their surface, and these vesicles are thought to constitute the readily releasable pool. However, the mechanism by which vesicles associated with ribbons contribute to release is still a mystery, in part because it has been difficult to monitor the activity of ribbons in living cells. One idea is hat the ribbon serves as a scaffold along which vesicles travel to the plasma membrane at the active zone, where they then dock and fuse. Another possibility is that ribbons facilitate compound exocytosis, by providing a means for vesicles to dock and fuse with each other. One major goal of this proposal is to test these two alternative views of ribbon function, using high-resolution imaging of fluorescently labeled ribbons and synaptic vesicles in living cells. If labeld vesicles move along ribbons, that motion should be directly observable. The project will employ several approaches for visualizing vesicles, including activity-dependent uptake of FM dyes, single-molecule tracking of vesicle proteins fused to photoactivatable variants of red fluorescent protein, and electron microscopy. In addition, vesicles labeled with a fluorescent reporter of exocytosis will allow localization of the site of vesicle fusion, to determine if vesicles fuse wit each other distal to the plasma membrane, or exclusively at the plasma membrane itself. Another major goal of the project is to determine the functional roles of proteins and physiological systems that dictate the dynamics of vesicle trafficking at the active zone, in order to arrive at a comprehensive understanding of the molecular mechanisms controlling neurotransmission at ribbon synapses. The results of the project will significantly advance fundamental knowledge about the early steps in transmission of sensory information in both vision and hearing.